Mixed-function oxidation of Escherichia coli glutamine synthetase results in the modification of a single histidine residue per subunit, introduction of a carbonyl group, and loss of catalytic activity. This oxidative inactivation results in increased susceptibility to proteolytic attack. Four proteases which preferentially degrade the oxidized form of [14C]-labeled glutamine synthetase have been purified from liver. One of the proteases was identified as cathepsin D which is of lysosomal origin. The other three proteases were present in the cytosol. Two of them required calcium for activity; the other did not. The latter protease, a thiol protease referred to simply as the alkaline protease, has been purified to apparent homogeneity from mouse and rat liver acetone powders. Its molecular weight, determined by gel filtration, was around 270,000. Multiple bands of molecular weight 25,000-32,000 were obtained on SDS gels. Native glutamine synthetase was not significantly degraded by the cytosolic proteases at physiological pH values. Oxidative inactivation of the enzyme caused a very dramatic increase in susceptability to attack by these proteases. Adenylylation, which causes reversible loss of glutamine synthetase biosynthetic activity, had little effect on its rate of degradation by any of the proteases. Proteolysis was increased following relaxation or dissociation of the enzyme. The characteristics of the oxidative modification of rabbit muscle enolase are similar to those of E. coli gluatamine systhetase. The oxidized form of this enzyme was also preferentially degraded by the purified liver alkaline protease. The ability of cytosolic protease to specifically recognize the oxidation form of glutamine synthetase and enolase suggests that oxidative modification of proteins may be involved in intracellular protein turnover.